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A human-derived insecticidal gene and insecticidal peptide encoded thereby and application thereof

a technology of human-derived insecticidal gene and insecticidal peptide, applied in the field of genetic engineering and biological control, can solve the problems of significant decrease of total white blood cells (wbc) and hemoglobin (hgb) of animals

Active Publication Date: 2017-03-30
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about a new type of antibody that can kill insects. It was made by screening human gene banks and has been tested on a specific type of insect. The antibody is small and can be produced in large amounts. This new resource is important for developing new types of insecticides that can lower safety risks and reduce the use of chemical insecticides.

Problems solved by technology

However, following the application and generalization of transgenic Bt crops, its possible potential hazards in gene escape, change of microbial ecological structure of soil, drug resistance of species and harm to normal immune system have gradually aroused the attention of the society.
Meanwhile, long-term use of Bt toxin protein at a large dose may also result in significant decrease of total white blood cells (WBC) and hemoglobin (HGB) of animals.

Method used

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  • A human-derived insecticidal gene and insecticidal peptide encoded thereby and application thereof
  • A human-derived insecticidal gene and insecticidal peptide encoded thereby and application thereof

Examples

Experimental program
Comparison scheme
Effect test

embodiment 1

ticidal Peptide

[0053](1) Add 20 μl of humanized phage antibody library bacterium liquid to 200 ml of 2×TY-AG fluid medium, cultivate it at constant temperature of 37° C. till OD600 is 0.4, measure 50 ml of the bacterium liquid, add 1×1012 pfu of helper phage KM13 for superinfection, incubate the liquid at 37° C. for 30 minutes, then centrifuge it at 3300 g for 10 minutes, discard the supernate, use 100 ml of 2×TY-AKG fluid medium to resuspend the precipitate and cultivate it at 30° C. overnight; centrifuge it at 3300 g for 30 minutes next day, collect the supernate, add 20 ml of PEG / NaCl solution, keep it in ice bath for 1 h, then centrifuge it at 3300 g for 30 minutes and resuspend the precipitate by 4 ml of PBS; centrifuge the resuspension solution at 11600 g for 10 minutes. The supernate is amplified phage antibody library;

[0054](2) Use the amplified phage antibody library obtained in step 1 for four rounds of Panning: The screening method is positive and negative screening. Nega...

embodiment 2

ary Culture of F2

[0062]The supernate obtained through screening in Embodiment 1 and containing insecticidal peptide is transferred to 10 ml of 2×TY-AG fluid medium at a volume ratio of 1:100 and incubated at 37° C. for 2 h. 100 μl of helper phage KM13 with titer of 1012 is added for rescue, incubated at 30° C. for 2 h and centrifuged at 1800 g for 10 minutes. The supernate is removed. 2×TY-AK fluid medium is used to resuspend the precipitated bacteria. It is cultivated while being shaken at 30° C. with 250 rpm overnight. Next day it is centrifuged at 1800 g for 30 minutes. Its supernate is supernate containing F2 primary culture.

embodiment 3

tification of F2

(1) ELISA Detection Experiment of Competitive Inhibition

[0063]The experiment adopts 6 experimental groups and corresponding control groups. Solutions are prepared based on Table 1.

TABLE 1Preparation of solutions for ELISA detection experiment of competitiveinhibitionIrrelevant Anti-Id2 × TY fluidGroupF2single-chain antibodymediumExperimental group 1 5 μl45 μlControl group 1 5 μl45 μlExperimental group 210 μl40 μlControl group 210 μl40 μlExperimental group 320 μl30 μlControl group 320 μl30 μlExperimental group 430 μl20 μlControl group 430 μl20 μlExperimental group 540 μl10 μlControl group 540 μl10 μlExperimental group 650 μlControl group 650 μl

[0064]In Table 1, F2 is the supernate obtained in Embodiment 2 and containing F2 primary culture;[0065]Add 50 μl of 10 μg / ml anti-Cry2Aa polyclonal antibody to the solutions prepared in Table 1 respectively, incubate them at 37° C. for 2 h, add them to a 96-well plate coated with 2 μg / ml Cry2Aa toxin respectively (the 96-well pl...

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Abstract

The present invention discloses a human-derived insecticidal gene and insecticidal peptide encoded by the same and application thereof. The nucleotide sequence of the human-derived insecticidal gene is as represented by SEQ ID NO.1. The amino acid sequence of the insecticidal peptide encoded by this gene is as represented by SEQ ID NO.2. The insecticidal peptide may be expressed through prokaryotic system. The primary culture has binding activity to Cnaphalocrocis medinalis midgut peritrophic membrane specific receptor BBMV. It is obtained without animal immunization and has a short production cycle and a small amino acid sequence. It is suitable for in vitro mass production and may lower the safety risks resulting from wide use of existing Bt toxins and even might substitute Bt to biologically control agricultural pests in the future. It has important scientific and practical significance to reducing the use of insecticides.

Description

FIELD OF THE INVENTION[0001]The present invention relates to genetic engineering and biological control field, particularly to a human-derived insecticidal gene and insecticidal peptide encoded by the same and application thereof.BACKGROUND OF THE INVENTION[0002]Currently, the insecticidal gene widely used in the world for biological control of pests is Bt toxin gene of Bacillus thuringiensis (Bt) (such as: Cry1C, Cry1Ab, Cry1B, Cry1F and Cry2Aa, etc.). Bacillus thuringiensis is insect pathogenic bacterium. The Bt toxin generated by Bacillus thuringiensis has a specific killing effect to many species of agricultural and forestry pests. Since Belgian Plant Genetic Systems first reported the success of transgenic Bt insecticidal tobacco in 1987 till today, Bt gene has been transferred to main crops in the world, such as: maize, paddy, cotton, tomato, potato and tobacco. According to the statistics of International Service for the Acquisition of Agri-biotech Applications (ISAAA) in 201...

Claims

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Application Information

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IPC IPC(8): C07K16/42C07K16/12A01N63/02A01N63/50
CPCC07K16/4233A01N63/02C07K2317/21C07K2317/622C07K2317/73C07K16/1278C07K14/47C12N15/63A01N47/44C12N15/8286Y02A40/146A01N63/50A01N63/10
Inventor LIU, XIANJINLIU, YUANXIE, YAJINGWU, AIHUAZHANG, XIAOXU, CHONGXINZHAO, YANYANZHONG, JIANFENG
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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